We have been studying membrane-myofibril links in developing and mature muscles, with the objective of understanding myofibril assembly, maintenance, and force transmission. Our previous work has shown that N-RAP, a novel muscle specific LIM protein with homology to nebulin, plays an important role in these processes. That work led us to propose a novel model for the initiation of myofibril assembly, which envisions N-RAP as a membrane-associated organizing center for assembly of alpha-actinin and actin into premyofibril complexes. In this fiscal year, we tested this model by expressing deletion mutants of N-RAP as fusion proteins linked to green fluorescent protein (GFP) in cultured embryonic chick cardiomyocytes, an excellent model system for studying myofibril assembly. Deletion mutants missing the membrane-targeting N-RAP LIM domain or the Z-line targeting IB region disrupted myofibril assembly, effectively inhibiting alpha-actinin organization into myofibrillar Z-lines and actin organization into sarcomeric filaments. In contrast, the deletion mutant missing the actin-binding super repeats did not interfere with alpha-actinin assembly, but remained associated with mature Z-lines. While permitting normal Z-line assembly, this construct greatly disrupted the organization of sarcomeric actin filaments, and the degree of this disruption appeared to increase with increased incorporation of the mutant protein at the Z-line. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes. The results strongly support our molecular model for the initiation of myofibril assembly. We used yeast two-hybrid technology to identify filamin-2, Krp1 (also called sarcosin), and alpha-actinin as new N-RAP binding partners. In vitro, we detected significant alpha-actinin binding to N-RAP-IB and the N-RAP LIM domain, filamin binding to N-RAP super repeats, and Krp1 binding to N-RAP super repeats and N-RAP-IB. Filamin, alpha-actinin and N-RAP are concentrated at the intercalated disks of adult hearts. We observed N-RAP colocalization with alpha-actinin and filamin during myofibril assembly in cultured chick cardiomyocytes. All three components appear to be concentrated in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. Filamin, along with alpha-actinin, appears to remain as a Z-line component in mature sarcomeres, while N-RAP is not found in the mature structures. The results are consistent with the hypothesis that N-RAP functions as a membrane-associated organizing center for assembly of premyofibril complexes. Our previous work has also implicated N-RAP up-regulation in the pathology of dilated cardiomyopathy in mice. We sought to evaluate N-RAP as a candidate disease gene in human cardiomyopathies. Linkage identifies human chromosome 10q24-26 as a locus for dilated cardiomyopathy (DCM), a region including the N-RAP gene. In this study, no N-RAP mutations were identified in 50 DCM and hypertrophic cardiomyopathy patients, but several polymorphisms were detected. Immunofluorescence of biopsy samples showed that human N-RAP is concentrated at the cardiac intercalated disk, and no differences in tissue specificity or mobility on immunoblots were detected between mouse and human N-RAP. Sequence data and comparison to the gene databases show that murine and human N-RAPs have similar gene structure, containing 42 exons. Alternative splicing of one exon was detected, and PCR experiments showed that >90% of skeletal muscle transcripts contain the alternatively spliced exon, while this exon is not found in cardiac N-RAP.